ABSTRACT Zika virus (ZIKV) is linked to microcephaly and other birth defects, and thus the emerging epidemic of ZIKV in the Americas poses significant health threats. Despite the considerable impact of ZIKV infection on world-wide health, no antiviral therapies are available, and existing flavivirus vaccines are of limited utility. Our long-term goal is to obtain detailed structural and biochemical information regarding ZIKV replication and to use this information for the development of antiviral therapeutics and vaccines. In particular, the ZIKV RNA genome contains a stem loop A (SLA) at the 5'-end that acts as a promoter element for the viral polymerase NS5 to initiate RNA synthesis. Thus, the structural information on ZIKV SLA and its interaction with ZIKV NS5 will be essential to understand the mechanism of NS5-mediated initiation of RNA replication. Knowledge of the mechanism by which the polymerase recognizes the SLA promoter will additionally identify potential therapeutic targets, and suggest strategies for intervention against ZIKV replication. Our goals of the project are to characterize the interaction site between ZIKV NS5 and SLA using binding assays (Aim 1) and determine the structure of ZIKV SLA by a combination of our pRNA fusion technique and cryo-electron microscopy (Aim 2). Because SLA and NS5 interaction is essential for viral replication, RNA decoys that bind NS5 specifically may inhibit ZIKV replication. We will thus identify structural features in SLA that are conserved among flaviviruses, and design SLA decoys as potential ZIKV inhibitors.